Wasabisides A–E, Lignan Glycosides from the ... - ACS Publications

Article pubs.acs.org/jnp

Wasabisides A−E, Lignan Glycosides from the Roots of Wasabia japonica Chung Sub Kim,† Lalita Subedi,‡,§ Oh Wook Kwon,⊥ Hyun Bong Park,∥,¶ Sun Yeou Kim,‡,§ Sang Un Choi,# and Kang Ro Lee*,† †

Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, Suwon 16419, Republic of Korea Gachon Institute of Pharmaceutical Science, Gachon University, Incheon 21936, Republic of Korea § College of Pharmacy, Gachon University, #191, Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea ⊥ Natural F&P Corp., 152 Saemal-ro, Songpa-gu, Seoul 05802, Republic of Korea ∥ Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States ¶ Chemical Biology Institute, Yale University, West Haven, Connecticut 06516, United States # Korea Research Institute of Chemical Technology, Daejeon 34114, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Five new lignan glycosides, wasabisides A−E (1−5), and four known phenolic compounds (6−9), were isolated from the roots of Wasabia japonica. The chemical structures of the new compounds (1−5) were determined through spectroscopic analysis and chemical methods. All isolated compounds (1−9) were evaluated for their potential neuroprotective effects through induction of nerve growth factor in C6 glioma cells, for their effects on nitric oxide levels in lipopolysaccharidestimulated murine microglia BV2 cells, and for their cytotoxicity against four human tumor cell lines (A549, SK-OV-3, SK-MEL2, and BT549).

W

In a continuing search for bioactive constituents from Korean medicinal plants, five new lignan glycosides, wasabisides A−E (1−5), were isolated and characterized structurally, along with four known phenolic compounds (6−9) from the roots of W. japonica. The structures of the new compounds (1−5) were elucidated by NMR (1H and 13C NMR, 1H−1H COSY, HSQC, HMBC, and NOESY), HRMS, ECD, and chemical methods. All isolated compounds (1−9) were evaluated for their cytotoxicity, potential neuroprotective activity, and effects on nitric oxide (NO) levels. To the best of our knowledge, this is the first report of biologically active lignan derivatives from W. japonica.

asabi [Wasabia japonica (Miq.) Matsum., Brassicaceae] is a perennial herb cultivated in Korea and Japan. The paste of its roots has been used for a long time as a traditional Japanese pungent spice to garnish dishes such as sushi and sashimi.1,2 The characteristic pungent flavor of W. japonica is derived from several isothiocyanates, mainly allyl isothiocyanate. Although 6-methylsulfinylhexyl isothiocyanate and 6methylthiohexyl isothiocyanate are derivatives of allyl isothiocyanate present in W. japonica, they are not pungent.3 These isothiocyanate analogues are the major sulfur compounds in W. japonica and have various biological effects, including anticancer, anti-inflammatory, antimicrobial, antioxidant, and anti-blood-clotting activities.4 In addition, previous investigations on W. japonica led to reports of several phenylpropanoid glycosides and flavonoid glycosides with antioxidant activities.2,5 Since most previous research on W. japonica has focused on the biological activities of these isothiocyanate compounds, an investigation into another chemical class possessing biological activity in W. japonica was pursued, namely, the lignan glycoside constituents. © 2016 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Wasabiside A (1) was isolated as a colorless gum. The molecular formula was determined to be C26H32O12 from the [M + Na]+ ion peak obtained by positive-ion HRFABMS. The Received: June 25, 2016 Published: October 4, 2016 2652

DOI: 10.1021/acs.jnatprod.6b00582 J. Nat. Prod. 2016, 79, 2652−2657

Journal of Natural Products

Article

HMBC correlation of H-1′/C-4 confirmed the location of the glucopyranosyl unit to be at C-4 (Figure 2). NMR analysis of the 1H−1H COSY, HSQC, and HMBC data corroborated the planar structure of 2, and the absolute configuration of 2 was determined to be the same as that of 1 through hydrolysis and the observation of a positive Cotton effect at 233 nm in the ECD spectrum. Thus, the structure of 2 was shown to be (7R,8S,8S′)-7-hydroxymatairesinol 4-O-β-D-glucopyranoside. Wasabiside C (3) gave a molecular formula of C27H34O13 as established by HRFABMS. The 1H and 13C NMR data of 3 resembled those of 2, except for the signals of a symmetric 1,3,4,5-tetrasubstituted aromatic ring [δH 6.59 (2H, s); δC 154.3 (×2), 140.9, 135.4, and 104.7 (×2)] instead of those of a 1,3,4trisubstituted aromatic ring. Thus, 3 was observed to be a 5methoxy derivative of 2, which was confirmed by the HMBC correlations of OCH3-5/C-5 (Figure 2). 2D NMR data analysis was used to determine the planar structure and relative configuration of 3. The absolute configuration of this compound was corroborated as being the same as 1 and 2 by hydrolysis and ECD spectroscopic analysis. Thus, the structure of 3 was determined as (7R,8S,8S′)-7-hydroxy-5-methoxymatairesinol 4-O-β-D-glucopyranoside. Wasabiside D (4) was purified as a colorless gum. The molecular formula of this isolate was found to be the same as that of 2 (C26H32O12) from the HRFABMS. The NMR data of 4 were quite similar to those of 2, with the main differences being that the signals for the aromatic protons in 4 [δH 7.02 (1H, d, J = 8.2 Hz), 6.80 (1H, d, J = 1.7 Hz), 6.74 (1H, d, J = 8.2 Hz), 6.71 (1H, dd, J = 8.2, 1.7 Hz), 6.66 (1H, dd, J = 8.2, 1.9 Hz), and 6.65 (1H, d, J = 1.9 Hz)] were shifted slightly compared to those of 2 [δH 7.11 (1H, d, J = 8.3 Hz), 6.84 (1H, dd, J = 8.3, 2.0 Hz), 6.83 (1H, d, J = 8.3 Hz), 6.64 (1H, d, J = 8.0 Hz), 6.57 (1H, d, J = 2.0 Hz), and 6.45 (1H, dd, J = 8.0, 2.0 Hz)]. This indicated the glucopyranose unit in 4 is located at C-4′. Furthermore, the HMBC cross-peak of H-1′/C-4′ corroborated the location of the glucopyranose unit (Figure 2). The planar structure of 4 was determined through 2D NMR data analysis, and the relative and absolute configurations of 4 were confirmed as being the same as those of 1−3 through hydrolysis and ECD spectroscopic analysis. Thus, the structure of 4 was assigned as (7R,8S,8S′)-7-hydroxymatairesinol 4′-O-βD-glucopyranoside. Wasabiside E (5) was obtained as a colorless gum with a molecular formula of C26H30O12. The 1H and 13C NMR spectra of this compound resembled those of 1, but a quite downfieldshifted carbon signal at δC 198.1 was detected instead of the carbon signal at δC 83.8 (C-7). The occurrence of a carbonyl group at C-7 was confirmed through the HMBC cross-peaks of H-2, H-6, H-8, H-9, and H-8′ to C-7, and the location of the glucopyranosyl unit was corroborated by the HMBC correlation as being between H-1″ and C-4 (Figure 3). The β- and D-form of the glucopyranosyl unit in compound 5 were determined through the same method as described for 1. The relative configuration between H-8 and H-8′ was confirmed as trans by the NOESY cross-peaks of H-8/H-2′ and H-7′b (Figure 3). A negative Cotton effect at 233 nm was observed in the ECD spectrum of 5, indicating that the absolute configuration of this compound is 8R,8′R.11,12 Therefore, the structure of 5 was elucidated as (8R,8′R)-7-oxomatairesinol 4O-β-D-glucopyranoside. The four known compounds isolated (6−9) were identified as trans-p-coumaric acid (6),13 trans-ferulic acid (7),14 benzoic

Chart 1

1

H NMR spectrum of 1 showed the presence of two 1,3,4trisubstituted aromatic rings [δH 6.89 (1H, brs), 6.77 (2H, overlap), 6.70 (1H, d, J = 8.0 Hz), 6.70 (1H, d, J = 1.8 Hz), and 6.58 (1H, dd, J = 8.0, 1.8 Hz)], an oxygenated methine [δH 4.75 (1H, d, J = 6.1 Hz)], an oxygenated methylene [δH 4.16 (1H, dd, J = 9.0, 7.3 Hz) and 3.99 (1H, t, J = 9.0 Hz)], two methines [δH 3.03 (1H, m) and 2.79 (1H, m)], two methoxy groups [δH 3.85 (3H, s) and 3.82 (3H, s)], a methylene [δH 2.98 (1H, dd, J = 13.8, 5.6 Hz) and 2.91 (1H, dd, J = 13.8, 5.6 Hz)], and a glucopyranosyl unit [δH 4.37 (1H, d, J = 7.2 Hz), 3.69 (1H, dd, J = 11.8, 2.4 Hz), 3.58 (1H, dd, J = 11.8, 5.4 Hz), 3.33 (1H, overlap), 3.31 (2H, overlap), and 3.11 (1H, ddd, J = 9.1, 5.4, 2.4 Hz)]. The 13C NMR spectrum of 1 displayed 26 carbon signals including 12 aromatic carbons (from δC 111.9 to 149.0), a carbonyl carbon (δC 182.1), two oxygenated carbons (δC 83.8 and 69.8), three methylene and methine carbons (δC 45.8, 44.9, and 35.8), two methoxy carbons [δC 56.5 (×2)], and a group of glucopyranose carbons (δC 104.2, 78.6, 78.0, 76.0, 71.5, and 62.8). These 1H and 13C NMR data of 1 (Table 1) were similar to those of (−)-7(S)-hydroxymatairesinol,6 except for the presence of the glucopyranose signals (see above). The planar structure of 1 was determined through 2D NMR analysis, including 1H−1H COSY, HSQC, and HMBC spectra. The HMBC cross-peak of H-7/C-1″ indicated that the glucopyranosyl unit is located at C-7 (Figure 1), and the coupling constant of the anomeric proton (J = 7.2 Hz) confirmed it as being in the β-form.7−10 Acid hydrolysis of 1 afforded Dglucopyranose, which was identified by its specific rotation 7−10 {[α]25 The D +60.5 (c 0.01, H2O)} and GC/MS analysis. relative configuration of 1 was elucidated from NOESY correlations and enzymatic hydrolysis. In the NOESY spectrum of 1, strong and weak correlations of H-8/H-9b and H-9a, respectively, and a correlation of H-9a/H-8′ were observed, which confirmed that H-8 and H-8′ are in the trans form, with both C-8 and C-8′ assigned as R* (Figure 1). A relatively small coupling constant value (6.6 Hz) between H-7 and H-8 of 1a, the hydrolysis product of 1, was identical to that of (−)-7(S)hydroxymatairesinol (7S,8R,8′R; 6.6 Hz), rather than (−)-7(R)-hydroxymatairesinol (7R,8R,8′R; 7.8 Hz), which corroborated the relative configuration of 1 as 7S*,8R*,8′R*.6 In the ECD spectrum of 1, a positive Cotton effect at 233 nm was observed, indicating the absolute configuration of 1 to be 8S,8′S.11,12 Thus, the structure of 1 was established as (7R,8S,8S′)-7-hydroxymatairesinol 7-O-β-D-glucopyranoside. Wasabiside B (2) was obtained as a colorless gum with the same molecular formula as 1 (C26H32O12). Inspection of the NMR data of 2 indicated that this compound is structurally quite similar to 1, with the major difference being an upfieldshifted carbon signal at C-7 (δC 74.7, 2; δC 83.8, 1), suggesting that the glucopyranosyl unit is at a location other than C-7. The 2653

DOI: 10.1021/acs.jnatprod.6b00582 J. Nat. Prod. 2016, 79, 2652−2657

2654

56.5 104.2 76.0 78.6 71.5 78.0 62.8

44.9 182.1 56.5

130.7 114.5 149.0 146.5 116.2 123.5 35.8

δC

132.3 111.9 149.0 147.6 115.9 121.1 83.8 45.8 69.8

position

1 2 3 4 5 6 7 8 9a 9b 1′ 2′ 3′ 4′ 5′ 6′ 7′a 7′b 8′ 9′ OCH3-3 OCH3-5 OCH3-3′ 1″ 2″ 3″ 4″ 5″ 6″a 6″b

overlap overlap d (6.1) m dd (9.0, 7.3) t (9.0)

δH

d (8.0) dd (8.0, 1.8) dd (13.8, 5.6) dd (13.8, 5.6) m

3.82, 4.37, 3.31, 3.33, 3.31, 3.11, 3.69, 3.58,

s d (7.2) overlap overlap overlap ddd (9.1, 5.4, 2.4) dd (11.8, 2.4) dd (11.8, 5.4)

3.85, s

6.70, 6.58, 2.98, 2.91, 3.03,

6.70, d (1.8)

6.77, 6.77, 4.75, 2.79, 4.16, 3.99,

6.89, brs

1 δC

56.5 102.9 75.1 78.0 71.5 78.3 62.7

44.2 182.5 56.7

130.7 114.1 148.9 146.4 116.2 123.2 36.4

138.8 111.4 150.8 147.4 117.5 119.6 74.7 46.7 71.1

δH

d (8.3) dd (8.3, 2.0) d (4.7) m overlap

d (8.0) dd (8.0, 2.0) dd (13.5, 5.2) overlap overlap

3.78, 4.93, 3.52, 3.50, 3.42, 3.44, 3.91, 3.73,

s d (7.5) overlap overlap overlap overlap dd (12.1, 2.0) dd (12.1, 5.2)

3.82, s

6.64, 6.45, 2.85, 2.76, 2.90,

6.57, d (2.0)

7.11, 6.84, 4.72, 2.65, 4.16,

6.83, d (2.0)

2 δC

43.9 182.5 57.0 57.0 56.4 105.5 75.9 77.9 71.4 78.4 62.7

130.6 114.0 148.9 146.4 116.2 123.2 36.3

140.9 104.7 154.3 135.4 154.3 104.7 74.5 46.7 71.2 s d (4.3) m overlap

δH

3.83, 3.83, 3.78, 4.90, 3.47, 3.51, 3.45, 3.28, 3.79, 3.69,

6.62, 6.36, 2.83, 2.74, 2.91, s s s d (7.5) overlap overlap overlap ddd (9.3, 5.0, 2.3) dd (12.1, 2.3) dd (12.1, 5.0)

d (8.0) dd (8.0, 1.8) dd (13.8, 7.1) dd (13.8, 5.3) overlap

6.60, d (1.8)

6.59, 4.72, 2.64, 4.23,

6.59, s

3

Table 1. 1H [ppm, mult., (J in Hz)] and 13C NMR Spectroscopic Data of Compounds 1−5 in CD3OD δC

56.7 103.0 75.0 78.0 71.5 78.3 62.7

44.3 182.3 56.5

134.2 114.9 150.7 146.9 117.6 123.4 36.3

135.6 110.7 149.1 147.1 116.1 119.7 75.0 46.9 70.9

δH

d (8.2) dd (8.2, 1.7) d (5.1) m dd (9.2, 6.2) t (8.7)

d (8.2) dd (8.2, 1.9) dd (13.3, 6.6) dd (13.3, 5.0) m

3.80, 4.89, 3.51, 3.49, 3.41, 3.43, 3.90, 3.71,

s d (7.3) overlap overlap overlap ddd (9.7, 5.3, 2.1) dd (12.1, 2.1) dd (12.1, 5.3)

3.82, s

7.02, 6.66, 2.92, 2.81, 2.94,

6.65, d (1.9)

6.74, 6.71, 4.67, 2.62, 4.16, 4.11,

6.80, d (1.7)

4 δC

56.3 102.0 74.9 78.0 71.4 78.5 62.6

47.5 180.0 56.8

130.4 113.9 149.1 146.6 116.4 123.1 36.3

132.0 112.6 150.9 153.0 116.1 124.3 198.1 48.5 70.3

δH

d (8.0) dd (8.0, 1.3) dd (14.0, 4.9) dd (14.0, 9.6) overlap

3.69, 5.04, 3.55, 3.52, 3.43, 3.52, 3.95, 3.74,

s d (7.6) dd (9.2, 7.6) overlap overlap overlap dd (12.1, 2.2) dd (12.1, 5.8)

3.88, s

6.59, 6.58, 3.16, 2.78, 3.44,

6.68, d (1.3)

4.35, q (8.2) 4.56, dd (8.7, 8.2) 4.16, dd (8.7, 8.2)

7.14, d (8.5) 7.29, dd (8.5, 2.1)

7.32, d (2.1)

5

Journal of Natural Products Article

DOI: 10.1021/acs.jnatprod.6b00582 J. Nat. Prod. 2016, 79, 2652−2657

Journal of Natural Products

Article

Figure 3. 1H−1H COSY (bold), HMBC (plain arrow), and NOESY (dashed) correlations of compound 5. Figure 1. 1H−1H COSY (bold), HMBC (plain arrows), and NOESY (dashed) correlations of compound 1.

Table 2. Effects of Compounds 1−9 on NGF Secretion in C6 Cells

acid (8),15 and syringic acid (9)16 by comparison with NMR and MS data in the literature. Many phenolic compounds were reported to be associated with neuroprotective activity, as measured by secretion of nerve growth factor (NGF) from C6 glioma cells in a previous study.7−11,17,18 Therefore, the NGF secretion effects were tested for compounds 1−9 using an enzyme-linked immunosorbent assay (ELISA) development kit. NGF release was measured into the medium and cell viability with an MTT assay. As shown in Table 2, compound 1 induced NGF secretion moderately by 150.7%, without producing a cytotoxic effect at 20 μM. The other compounds evaluated showed weak activity (99.4−129.2%). To investigate the effect of the isolated compounds (1−9) on neuroinflammation, NO levels were measured in murine microglia BV2 cells stimulated with lipopolysaccharide (LPS). However, none of the compounds showed any activity (IC50 > 50 μM) in this assay. The cytotoxic activities of compounds 1−9 were evaluated against the A549 (non-small-cell lung adenocarcinoma), SKOV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and BT549 (invasive ductal carcinoma) cell lines using the sulforhodamine B (SRB) bioassay. Compound 6, trans-pcoumaric acid, showed cytotoxic activity against the BT549 cell line, with an IC50 value of 10 μM. The other compounds tested were inactive (IC50 > 10 μM) for all cancer cell lines used.

■

compound

NGF secretiona (%)

1 2 3 4 5 6 7 8 9 6-shogaolc

150.7 103.3 99.4 104.3 122.1 129.2 110.7 114.5 125.5 165.7

cell viabilityb (%) 100.4 98.2 101.6 102.3 100.9 198.0 95.0 96.4 95.9 115.3

± ± ± ± ± ± ± ± ± ±

2.55 3.47 2.71 6.73 7.21 4.83 0.91 1.98 3.49 6.32

a C6 cells were treated with 20 μM of each compound. After 24 h, the content of NGF secreted in the C6-conditioned medium was measured by ELISA. The level of secreted NGF is expressed as the percentage of the untreated control (set as 100%). bCell viability after treatment with 20 μM of each compound was determined by an MTT assay and is expressed as a percentage (%). Results are the means of three independent experiments, and the data are expressed as mean ± SD. cPositive control substance.

Gilson 306 pump (Middleton, WI, USA) with a Shodex refractive index detector (New York, NY, USA). Column chromatography was performed with silica gel 60 (70−230 and 230−400 mesh; Merck, Darmstadt, Germany) and RP-C18 silica gel (Merck, 230−400 mesh). Merck precoated silica gel F254 plates and RP-18 F254s plates (Merck) were used for thin-layer chromatography (TLC). Spots were detected on TLC under UV light or by heating after spraying the samples with anisaldehyde-sulfuric acid. Plant Material. The roots of W. japonica (3.3 kg) were collected in Hanam, Republic of Korea, in October 2014. The plant was identified by one of the authors (K.R.L.). A voucher specimen (SKKU-NPL 1409) of the plant has been deposited at the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea. Extraction and Isolation. The roots of W. japonica (3.3 kg) were extracted with 80% aqueous MeOH at room temperature and filtered. The filtrate was evaporated under reduced pressure to obtain the MeOH extract (750 g), which was suspended in distilled H2O and successively partitioned with hexane, CHCl3, EtOAc, and n-BuOH, yielding 1.3, 5.6, 5.8, and 2.7 g of each residue, respectively. The EtOAc-soluble fraction (5.8 g) was separated by passage over Diaion HP-20 resin by elution with MeOH−H2O (0:1, 1:4, 2:3, 3:2, 4:1, and

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter (JASCO, Easton, MD, USA). IR spectra were recorded on a Bruker IFS-66/S Fouriertransform IR spectrometer (Bruker, Karlsruhe, Germany). UV spectra were recorded with a Shimadzu UV-1601 UV−visible spectrophotometer (Shimadzu, Tokyo, Japan). ECD spectra were recorded with a JASCO J-810 spectropolarimeter (JASCO, Tokyo, Japan). NMR spectra were recorded on a Bruker AVANCE III 700 NMR spectrometer at 700 MHz (1H) and 175 MHz (13C). HRFABMS and HRESIMS were measured on either a Waters SYNAPT G2 (Milford, MA, USA) or a JEOL JMS700 mass spectrometer (JEOL, Peabody, MA, USA). The semipreparative HPLC system used had a

Figure 2. Key HMBC (plain arrows) correlations of compounds 2−4. 2655

DOI: 10.1021/acs.jnatprod.6b00582 J. Nat. Prod. 2016, 79, 2652−2657

Journal of Natural Products

Article

with cellulase (20 mg, from Aspergillus niger; ICN Biomedicals, Inc.) at 37 °C for 24 h. Each reaction mixture was extracted with EtOAc to yield 0.3−0.5 mg of 1a (from 1, 2, and 4), 3a (from 3), and 5a (from 5). (−)-7(S)-Hydroxymatairesinol (1a): colorless gum; [α]25 D −15.0 (c 0.01, CHCl3); 1H NMR (CDCl3, 700 MHz) δ 6.89 (1H, d, J = 8.1 Hz), 6.81 (1H, d, J = 7.7 Hz), 6.75 (1H, dd, J = 8.1, 1.7 Hz), 6.70 (1H, d, J = 1.6 Hz), 6.63 (2H, overlap), 5.67 (1H, s), 5.55 (1H, s), 4.66 (1H, d, J = 6.6 Hz), 3.97 (2H, overlap), 3.88 (3H, s), 3.84 (3H, s), 3.05 (1H, m), 2.95 (2H, overlap), 2.63 (1H, m); FABMS (positive-ion mode) m/z 375.1 [M + H]+. (−)-7(S)-Hydroxy-5-methoxymatairesinol (3a): colorless gum; 1 [α]25 D −32.0 (c 0.01, CHCl3); H NMR (CD3OD, 700 MHz) δ 6.61 (1H, d, J = 7.9 Hz), 6.49 (1H, d, J = 2.0 Hz), 6.48 (2H, s), 6.42 (1H, dd, J = 7.9, 2.0 Hz), 4.65 (1H, d, J = 4.7 Hz), 4.16 (1H, dd, J = 9.1, 5.8 Hz), 4.12 (1H, dd, J = 9.1, 8.1 Hz) 3.78 (6H, s), 3.72 (3H, s) 2.87 (1H, dt, J = 6.9, 5.6 Hz), 2.80 (1H, dd, J = 13.7, 6.9 Hz), 2.73 (1H, dd, J = 13.7, 5.5 Hz), 2.58 (1H, m); FABMS (positive-ion mode) m/z 405.1 [M + H]+. (+)-7-Oxomatairesinol (5a): colorless gum; [α]25 D +37.0 (c 0.01, CHCl3); 1H NMR (CDCl3, 700 MHz) δ 7.38 (1H, d, J = 2.1 Hz), 7.26 (1H, dd, J = 8.4, 2.2 Hz), 6.85 (1H, d, J = 8.4 Hz), 6.76 (1H, d, J = 8.0 Hz), 6.65 (1H, d, J = 1.9 Hz), 6.58 (1H, dd, J = 8.0, 1.8 Hz), 5.52 (s, 1H), 4.41 (1H, m), 4.13 (2H, overlap), 3.94 (3H, s), 3.78 (3H, s), 3.53 (1H, m), 3.04 (2H, overlap); FABMS (positive-ion mode) m/z 373.1 [M + H]+. Acid Hydrolysis of Compounds 1−5 and Sugar Analysis. Compounds 1−5 (each 0.5−1.0 mg) were refluxed with 1 mL of 1 N HCl for 1 h at 100 °C. The hydrolysate was extracted with EtOAc, and the aqueous layer was neutralized by passage through an Amberlite IRA-67 column (Rohm and Haas) and was repeatedly evaporated to give D-glucopyranose {[α]25 D +60.5 (c 0.05, H2O)}, which was detected by co-injection of the hydrolysate with standard silylated samples, giving a single peak at 9.721 min by GC-MS analysis under the following conditions: capillary column, HP-5MS UI (30 m × 0.25 mm × 0.25 μm, Agilent), column temperature, 230 °C; injection temperature, 250 °C; carrier gas, N2. An authentic sample of Dglucopyranose (Sigma) treated in the same way showed a single peak at 9.726 min. NGF and Cell Viability Assays. C6 glioma cells (purchased from the Korean Cell Line Bank, Seoul, Korea) were used to measure the release of NGF into the culture medium. C6 cells were seeded onto 24-well plates at a density of 1 × 105 cells/well, and, after 24 h, the cells were treated with serum-free DMEM and different concentrations of compound for an additional 24 h. The medium supernatant was collected from the culture plates, and NGF levels were measured using an ELISA development kit. Cell viability was measured using a 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The results are expressed as a percentage of the control group (untreated cells). 6-Shogaol was used as the positive control. Measurement of NO Production and Cell Viability in LPSActivated BV-2 Cells. The inhibitory effect of the test compounds on LPS-stimulated NO production was studied using BV2 cells. BV2 cells were originally developed by Dr. V. Bocchini at the University of Pergia (Pergia, Italy). The cells were seeded on a 96-well plate (4 × 104 cells/well) and treated with or without different concentrations of the compounds. These cells were stimulated with LPS (100 ng/mL) and incubated for 24 h. The concentration of nitrite (NO2), a soluble oxidation product of NO, in the culture medium was measured using Gries reagent (0.1% N-1-naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). Fifty microliters of supernatant was mixed with an equal volume of Gries reagent. Absorbance was measured after 10 min using a microplate reader (Emax, Molecular Devices, Sunnyvale, CA, USA) at 570 nm wavelength. NG-Monomethyl-L-arginine, a well-known nitric oxide synthase inhibitor, was used as a positive control (IC50 17.6 μM). Graded sodium nitrite solution was used as a standard to calculate nitrite concentrations. Cell viability was evaluated by observing the ability of viable cells to reduce the yellow-colored MTT to purplecolored formazan in the MTT assay.

1:0), to give six fractions (E1−E6). Fraction E4 (200 mg) was separated over a silica gel column (CHCl3−MeOH−H2O, 3:1:0.15) to yield nine subfractions (E41−E49). Subfraction E43 (25 mg) was purified by semipreparative HPLC (2 mL/min, 25% aqueous MeCN) to give compound 6 (2 mg). Subfraction E46 (40 mg) was purified by semipreparative HPLC (CHCl3−MeOH−H2O, 6:1:0.1) to give compound 1 (10 mg). Fraction E5 (160 mg) was chromatographed on a silica gel column (CHCl3−MeOH−H2O, 3:1:0.15) to yield five subfractions (E51−E55). Compounds 7 (2 mg), 8 (2 mg), and 9 (2 mg) were obtained by purifying subfraction E52 (17 mg) using semipreparative HPLC (45% aqueous MeOH). Subfraction E55 (13 mg) was purified by semipreparative HPLC (45% aqueous MeOH) to give compound 5 (4 mg). The n-BuOH-soluble fraction (2.7 g) was separated over Diaion HP-20 resin with MeOH−H2O (0:1 and 1:0) and further purified over a silica gel column (CHCl3−MeOH−H2O, 3:1:0.1) to yield 10 subfractions (B1−B10). Subfraction B3 (250 mg) was subjected to passage over a Lobar-A RP-C18 column with 40% aqueous MeOH and further purified by semipreparative HPLC (EtOAc−MeOH−H2O, 7:1:0.1) to give compounds 2 (6 mg) and 3 (7 mg). Subfraction B4 (300 mg) was separated using a Lobar-A RPC18 column with 20% aqueous MeOH and further purified by semipreparative HPLC (35% aqueous MeOH) to give compound 4 (1 mg). Wasabiside A (1): colorless gum; [α]25 D +9.6 (c 0.5, MeOH); IR (KBr) νmax 3358, 2945, 2832, 1452, 1033 cm−1; UV (MeOH) λmax (log ε) 280 (1.11), 232 (2.17) nm; CD (MeOH) λmax (Δε) 282 (−2.29), 233 (+6.91), 214 (−2.77) nm; 1H (700 MHz) and 13C (175 MHz) NMR data in CD3OD, see Table 1; HRFABMS (positive-ion mode) m/z 559.1789 [M + Na]+ (calcd for C26H32O12Na, 559.1791). Wasabiside B (2): colorless gum; [α]25 D +30.4 (c 0.01, MeOH); IR (KBr) νmax 3360, 2955, 2830, 1460, 1030 cm−1; UV (MeOH) λmax (log ε) 284 (1.17), 231 (2.14) nm; CD (MeOH) λmax (Δε) 281 (−2.18), 233 (+6.83), 213 (−2.75) nm; 1H (700 MHz) and 13C (175 MHz) NMR data in CD3OD, see Table 1; HRFABMS (positive-ion mode) m/z 559.1792 [M + Na]+ (calcd for C26H32O12Na, 559.1791). Wasabiside C (3): colorless gum; [α]25 D +10.3 (c 0.3, MeOH); IR (KBr) νmax 3358, 2948, 2831, 1454, 1035 cm−1; UV (MeOH) λmax (log ε) 280 (1.15), 234 (2.20) nm; CD (MeOH) λmax (Δε) 284 (−2.25), 234 (+6.61), 214 (−2.87) nm; 1H (700 MHz) and 13C (175 MHz) NMR data in CD3OD, see Table 1; 1H NMR (pyridine-d5, 700 MHz) δ 7.65 (1H, d, J = 3.3 Hz, OH-7), 7.17 (1H, d, J = 8.0 Hz, H-5′), 7.02 (1H, d, J = 1.4 Hz, H-2′), 6.97 (2H, s, H-2 and H-6), 6.87 (1H, dd, J = 8.0, 1.4 Hz, H-6′), 5.82 (1H, d, J = 6.7, H-1″), 5.03 (1H, t, J = 4.0 Hz, H-7), 4.41 (1H, dd, J = 12.0, 2.0 Hz, H-6″a), 4.35 (1H, overlap, H-2″), 4.34 (3H, overlap, H-9a, H-4″ and H-5″), 4.33 (1H, overlap, H-6″b), 4.17 (1H, t, J = 8.6 Hz, H-9b), 3.97 (1H, m, H-3″), 3.78 (6H, s, OCH3-3 and OCH3-5), 3.77 (3H, s, OCH3-3′), 3.44 (1H, m, H-8′), 3.30 (1H, dd, J = 13.8, 5.5 Hz, H-7′a), 3.08 (1H, dd, J = 13.8, 5.7 Hz, H-7′b), 2.97 (1H, m, H-8); 13C NMR (pyridine-d5, 175 MHz) δ 180.4 (C-9′), 154.3 (C-3 and C-5), 149.0 (C-3′), 147.4 (C-4′) 140.6 (C-1), 135.7 (C-4), 129.9 (C-1′), 123.6 (C-5′), 116.8 (C-6′), 114.6 (C-2′), 105.5 (C-1″), 105.2 (C-2 and C-6), 79.2 (C-3″), 78.9 (C-5″), 76.6 (C2″), 74.3 (C-7), 72.1 (C-4″), 69.9 (C-9), 63.1 (C-6″), 57.0 (OCH3-3 and OCH3-5), 56.3 (OCH3-3′), 46.4 (C-8), 43.8 (C-8′), 35.9 (C-7′); HRFABMS (positive-ion mode) m/z 589.1899 [M + Na]+ (calcd for C27H34O13Na, 589.1897). Wasabiside D (4): colorless gum; [α]25 D +57.7 (c 0.5, MeOH); IR (KBr) νmax 3362, 2952, 2832, 1451, 1035 cm−1; UV (MeOH) λmax (log ε) 280 (1.12), 232 (2.20) nm; CD (MeOH) λmax (Δε) 282 (−2.30), 233 (+6.99), 212 (−2.71) nm; 1H (700 MHz) and 13C (175 MHz) NMR data in CD3OD, see Table 1; HRFABMS (positive-ion mode) m/z 559.1790 [M + Na]+ (calcd for C26H32O12Na, 559.1791). Wasabiside E (5): colorless gum; [α]25 D +70.0 (c 0.003, MeOH); IR (KBr) νmax 3401, 2941, 2832, 1613, 1449 cm−1; UV (MeOH) λmax (log ε) 274 (1.16), 230 (2.73) nm; CD (MeOH) λmax (Δε) 316 (+2.55), 274 (−2.45), 233 (−3.87), 217 (+4.79) nm; 1H (700 MHz) and 13C (175 MHz) NMR data in CD3OD, see Table 1; HRESIMS (positiveion mode) m/z 535.1812 [M + H]+ (calcd for C26H31O12, 535.1816). Enzymatic Hydrolysis of Compounds 1−5. A solution of each sample (0.5−1.0 mg) in H2O (1.5 mL) was individually hydrolyzed 2656

DOI: 10.1021/acs.jnatprod.6b00582 J. Nat. Prod. 2016, 79, 2652−2657

Journal of Natural Products

Article

Cytotoxicity Assessment. The cytotoxicity of the compounds against the cultured human tumor cell lines A549, SK-OV-3, SK-MEL2, and BT549 was evaluated by the SRB method.19 All the cells tested were purchased from the American Type Culture Collection (Manassas, VA, USA) and maintained at the Korea Research Institute of Chemical Technology. Cisplatin was used as the positive control. This compound exhibited IC50 values of 1.12, 1.82, 1.27, and 1.25 μM against the A549, SK-OV-3, SK-MEL-2, and BT549 cell lines, respectively.

■

(17) Kim, C. S.; Subedi, L.; Kwon, O. K.; Kim, S. Y.; Yeo, E.-J.; Choi, S. U.; Lee, K. R. Bioorg. Med. Chem. Lett. 2016, 26, 351−354. (18) Kim, C. S.; Subedi, L.; Kim, S. Y.; Choi, S. U.; Kim, K. H.; Lee, K. R. J. Nat. Prod. 2015, 78, 1174−1178. (19) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00582. 1D and 2D NMR data of 1−5; CD data of 1 and 5 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Tel (K. R. Lee): 82-31-290-7710. Fax: 82-31-290-7730. Email: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Postdoctoral Research Program of Sungkyunkwan University (2015). We are thankful to the Korea Basic Science Institute (KBSI) for the measurements of mass spectra.

■

REFERENCES

(1) Okamoto, T.; Akita, N.; Nagai, M.; Hayashi, T.; Suzuki, K. J. Nat. Med. 2014, 68, 144−153. (2) Hosoya, T.; Yun, Y. S.; Kunugi, A. Phytochemistry 2008, 69, 827− 832. (3) Uchida, K.; Miura, Y.; Nagai, M.; Tominaga, M. Chem. Senses 2012, 37, 809−818. (4) Wu, S.-H.; Chyau, C.-C.; Chen, J.-H.; Tu, S.-F.; Lin, H.-H.; Chou, F.-P. J. Funct. Foods 2015, 14, 445−455. (5) Hosoya, T.; Yun, Y. S.; Kunugi, A. Tetrahedron 2005, 61, 7037− 7044. (6) Fischer, J.; Reynolds, A. J.; Sharp, L. A.; Sherburn, M. S. Org. Lett. 2004, 6, 1345−1348. (7) Kim, C. S.; Subedi, L.; Park, K. J.; Kim, S. Y.; Choi, S. U.; Kim, K. H.; Lee, K. R. Fitoterapia 2015, 106, 147−152. (8) Kim, C. S.; Subedi, L.; Kim, S. Y.; Choi, S. U.; Choi, S. Z.; Son, M. W.; Kim, K. H.; Lee, K. R. Phytochem. Lett. 2015, 14, 215−220. (9) Kim, C. S.; Kwon, O. W.; Kim, S. Y.; Lee, K. R. J. Braz. Chem. Soc. 2014, 25, 907−912. (10) Kim, C. S.; Kwon, O. W.; Kim, S. Y.; Kim, K. H.; Lee, K. R. Heterocycles 2014, 89, 1913−1922. (11) Kim, C. S.; Kwon, O. W.; Kim, S. Y.; Lee, K. R. J. Nat. Prod. 2013, 76, 2131−2135. (12) Yang, Y.-N.; Huang, X.-Y.; Feng, Z.-M.; Jiang, J.-S.; Zhang, P.-C. J. Agric. Food Chem. 2014, 62, 9095−9102. (13) Salum, M. L.; Robles, C. J.; Erra-Balsells, R. Org. Lett. 2010, 12, 4808−4811. (14) Prachayasittikul, S.; Suphapong, S.; Worachartcheewan, A.; Lawung, R.; Ruchirawat, S.; Prachayasittikul, V. Molecules 2009, 14, 850−867. (15) Bernini, R.; Coratti, A.; Provenzano, G.; Fabrizi, G.; Tofani, D. Tetrahedron 2005, 61, 1821−1825. (16) Chiji, H.; Tanaka, S.; Izawa, M. Agric. Biol. Chem. 1980, 44, 205−207. 2657

DOI: 10.1021/acs.jnatprod.6b00582 J. Nat. Prod. 2016, 79, 2652−2657